Functional identification of unknown enzymes discovered in genome projects presents a major challenge for contemporary biology: at least 50% of the proteins for which sequences are available continue to have uncertain, unknown, or incorrectly assigned functions. Without these functions, the potential value of genome sequences cannot be realized. This Collaborative Project is focused on developing and applying an integrated sequence-structure- function strategy for computational prediction and experimental verification of both in vitro and in vivo functions (developed with the support of P01 GM071790, "Deciphering Enzyme Specificity"). The Project requests funds for Core Resources that will include 1) computational facilities for bioinformatics, homology modeling, and in silico ligand docking;2) protein production and structural determination by X-ray crystallography;and 3) chemical synthesis of focused libraries of known or potential metabolites. The Project also requests funds for Bridging Projects that will enable the 1) enhancements in capabilities for the chemical and/or enzymatic syntheses of novel classes of substrates, 2) development of a microbiology component to allow in vivo assessment of physiological functions, and 3) the expansion of the project to include the glutathione transferase and haloalkane dehalogenase superfamilies that include biomedically important enzymes. Finally, the Project also requests funds for Pilot Projects in areas such as the functions of enzymes in the biosynthetic pathways for complex, biomedically important, secondary metabolites. The participants include: J. A. GerIt (enolase superfamily, PI) and J. E. Cronan (microbiology), both at University of Illinois, Urbana-Champaign;S. C. Almo (structural biology) at Albert Einstein College of Medicine;P. C. Babbitt (bioinformatics), M. P. Jacobson (in silico ligand docking and homology modeling), A. Sali (homology modeling), and B. K. Shoichet (in silico ligand docking), all at the University of California, San Francisco;F. M. Raushel (amidohydrolase superfamily) at Texas A&M University;R. N. Armstrong (glutathione transferase superfamily) at Vanderbilt University School of Medicine, K. N. Allen (haloalkane dehalogenaes superfamily) at Boston University, D. Dunaway-Mariano (haloalkane dehalogenase superfamily) at University of New Mexico, and S. K. Burley (structural genomics) at SGX Pharmaceuticals. This group of investigators brings together the necessary range of expertise to establish an effective Enzyme Function Initiative. Relevance: The assignment of function to the complete set of proteins encoded by genomes is a major challenge. However, when solved, their roles in molecular, cellular, and organismal functions will be known and novel targets for specific small molecule intervention and new approaches for therapeutic design can be identified. This Large-Scale Collaborative Project is focused on developing and implementing an integrated sequence- structure-computation strategy for predicting the substrate specificities of unknown enzymes discovered in genome projects, including classes of proteins with direct relevance to human health.